The present invention relates generally to crystallization chambers, and more particularly, is directed to a device for forming crystals with vapor diffusion in the hanging drop method.
Supersaturated solutions of macromolecules (such as proteins and nucleic acids) and peptides under conditions of defined pH, temperature and precipitant levels form crystals. Macromolecular crystals have been used in the biotechnology-pharmaceutical industry for many purposes. For example, three-dimensional models of macromolecule structures derived from X-ray diffraction analysis of crystals are used to design new drugs in pharmaceutical research. As another example, crystallization steps are utilized in purification-manufacturing processes of biotechnology-derived products. Further, crystalline complexes such as zinc-insulin are used for controlled release drug formulations.
It is necessary, however, to produce the correct conditions for macromolecular crystallization. This requires screening a wide range of conditions. See, for example, A. McPherson, Preparation and Analysis of Protein Crystals, John Wiley and Sons, New York, N.Y., pages 82-127, 1982.
Various microtechniques are presently used to discover conditions for macromolecule crystallization, including, for example, the free interface diffusion method (see F. R. Salemme, Arch. Biochem. Biophys., pages 151 and 533, 1972), vapor diffusion in the hanging or sitting drop method (see A. McPherson, Preparation and Analysis of Protein Crystals, John Wiley and Sons, New York, N.Y., pages 96-97, 1982), and liquid dialysis (see K. Bailey, Nature, pages 145 and 934, 1940).
Of the presently used methods, vapor diffusion is the most commonly used method for growing macromolecular crystals from solution, and the most common technique used for screening conditions for crystallization is vapor diffusion in the hanging drop method. See R. H. Davies and D. M. Segal, Methods in Enzymology. Academic Press, New York, N.Y., Vol. 22, page 266, 1971. The vapor diffusion method has advantages over other crystallization methods because it is truly a micro-crystallization technique. Vapor diffusion in the hanging drop technique allows screening of a large range of conditions while utilizing a relatively small amount of macromolecule or peptide.
For the formation of crystals from a protein, the vapor diffusion hanging droplet method is known. Specifically, a droplet containing a macromolecular solution is suspended in a sealed chamber. The macromolecular solution in the droplet is allowed to equilibrate with a reservoir containing a higher concentration of precipitating agent. Over time, water vapor diffuses from the less concentrated macromolecular solution to the more concentrated reservoir solution and slowly increases the concentration of macromolecule and precipitating agent within the droplet.
As an example, in a sealed (gas and vapor impermeable) chamber, a reservoir of, for example, 1 ml of 10% saturated ammonium sulphate, is provided. On the inside wall of the cover of the system, a 10 .mu.l protein droplet of, for example, 5% saturated ammonium sulphate, is provided. Because of the difference in vapor pressure between the droplet and the reservoir, water will evaporate from the droplet until an equilibrium results. Thus, the droplet may shrink, for example, 50% from 10 .mu.l to 5 .mu.l, so that a crystal will form.
In particular, vapor diffusion in hanging drop experiments are typically performed in 24 well tissue culture plates of the type sold by Linbro Flow Laboratories of McLean, Va. (Linbro Tissue Culture Multiwell Plate/Cover, Catalog No. 76-033-05) and Becton Dickinson and Company of Lincoln Park, N.J. (under the trademark FALCON 3047 MULTIWELL). The reservoir solutions are placed within each of the 24 wells of the tissue culture plate. The rim of each well is then greased with a silicon grease, such as a high vacuum grease sold by Dow Corning Corporation of Midland, Mich. Micro coverglasses or cover slips, for example, having a No. 2 thickness and an 18 mm diameter, are siliconized with siliconizing agents, such as a siliconizing agent sold under the trademark SURFASIL by Pierce Chemical Company of Rockford, Ill. A 1-40 .mu.l droplet containing a concentrated buffered solution of a homogeneous macromolecule and a precipitating agent, such as saturated ammonium sulfate, polyethylene glycol polymer, or a low molecular weight alcohol or solvent, is dispensed on each siliconized coverglass. The coverglasses are then inverted over the greased wells of the tissue culture plate and sealed by the silicon grease thereon. Typically, several components, such as buffers, salts, macromolecule concentration and precipitating agents, of both the droplet and reservoir solution in the wells are systematically varied, as well as conditions of vapor pressure, temperature, concentrations and the like.
With the coverglasses inverted, each droplet hangs down from its respective coverglass over or adjacent to the respective reservoir. Each experiment is generally allowed to equilibrate under 4.degree. C. or 22.degree. C. incubation conditions, and is monitored microscopically for crystal growth over various time intervals, for example, 3 days, 7 days, 1 month and 3 months, although other time intervals can be used.
Typically, several thousand experiments must be performed before conditions are found to produce high quality crystals. In this regard, it is noted that the setup of vapor diffusion hanging drop experiments is a very labor-intensive process which must be performed by experienced technical personnel. For example, multiple aspirating and dispensing steps of components, multiple greasing and polishing steps and the like must be performed in the experimental setup. Further, for each well, a separate cover slip must be manually inverted thereover. The volume and complexity of steps can produce a wide variation in experimental results. Still further, manpower restraints usually limit the range of conditions screened for crystallization.
For the above mentioned reasons, several research groups have developed their own automated crystallization systems.
The first robotic crystallization system based on the hanging drop method has been commercially available since 1987 from ICN Biomedicals. The system is a computer controlled sample preparation system, including a color monitor, a printer and a menu driven computer program. The system utilizes a 24 well multiwell plate and performs all of the required aspirating and dispensing steps of a classical coverglass-multiwell plate hanging drop setup. Specifically, the system includes means to automatically pipette reservoir solution into the wells, and to automatically pipette droplets onto the coverglasses or cover slips. However, this system requires full time intervention of a technician to manipulate the dispensed droplets on coverglasses over the corresponding wells. In other words, the technician must still manually apply silicon grease to the rim of each well, and then invert each coverglass over its respective well. This, of course, is time-consuming and burdensome.
A second approach to automated crystallization was developed at Lilly Research Laboratories in Indianapolis, Ind. in collaboration with the U.S. Naval Institute, and has been designated "APOCALYPSE", a fully automated system. See N. D. Jones et al, Annual Meeting of the American Crystallographic Association, page 27, 1987, and K. B. Ward et al, J. Crystal Growth, pages 90 and 325, 1988. The system utilizes a robot sold by Zymark Corporation under the trademark ZYMATE II, and a Master Laboratory pipetting station. In addition, the system uses a specially designed plate sold by Flow Laboratories under the trademark CRYSTALPLATE. The plate has a 3.times.5 array of wells for crystallization experiments. Each crystallization well has two coverglasses and two oil troughs to be filled. Specifically, there is a lower square-shaped oil trough surrounding a lower opening and an upper square-shaped oil trough surrounding an upper opening, the upper oil trough being larger than the lower oil trough. One coverglass is positioned over the lower oil trough so as to seal the lower opening and another coverglass is positioned over the upper oil trough so as to seal the upper opening. As a result, a sealed chamber is formed between the upper and lower coverglasses. A reservoir is formed adjacent the oil troughs and is in gaseous communication with the sealed chamber.
However, numerous operations are required to set up and seal each well. Specifically, oil must first be dispensed into each trough. Then, the reservoir must be filled. The lower cover slip is then positioned over the lower oil trough so as to seal the lower opening. A droplet is then deposited on the upper cover slip, which is subsequently inverted and positioned over the upper oil trough.
This specially designed plate has several advantages over the aforementioned classical coverglass-tissue culture plate set-up. First, the plate can be readily handled by a forklift hand of an articulated robotic arm such as the Zymark robot. The plate also has excellent optical visualization properties since the droplet is not viewed through a reservoir, that is, the reservoir is adjacent the droplet rather than beneath it. In addition, the plate can accommodate either hanging (from the upper coverglass), sitting (on the lower coverglass) or sandwiched (in contact with both coverglasses) drops.
However, the plate has many disadvantages. In the first place, there are cumbersome multiple coverglasses to be handled. Further, because the plate uses an oil trough to seal each well of, the coverglass-crystallization chamber, additional time must be spent ensuring the correct height of the oil in the troughs. In other words, the height of the oil has to be precise in order to obtain a meniscus which will ensure sealing of the coverglasses. For example, if the oil height is too low, there will be no seal. On the other hand, if the oil height is too high, the oil from the upper oil trough will run into the reservoir and/or lower oil trough, and the oil from the lower oil trough will run into the reservoir. Still further, the plate has a relatively slow equilibration rate compared to comparable classical coverglass-multiwell plate experiments. Lastly, conditions for crystallizing macromolecules in this plate have been found to be considerably different from conventional hanging drop experiments.
A third approach to automated crystallization has been developed by Cryschem Corporation using the "Biomek" automated liquid handling system. See D. Morris et al, Biotechniques, Vol. 7, No. 5, 1989. With this approach, a specially designed plate was developed and sold under the designation MD/24 for use in this automated system. The plate has 24 wells, each well having a center post or tee for standing drops and each well being surrounded by the reservoir at a slightly lower level than the center post but in gaseous communication therewith. In order to provide the sealed chambers, a clear mylar film from Corning Glass Co. is sealed over the plate. Thus, there is no coverglass manipulation involved in setting up experiments. Droplets are dispensed directly on the center tee and subsequently sealed with the mylar film.
There are several disadvantages with the MD/24 plate. First, the mylar film has poor optical properties. Further, in order to view experiments, the mylar film must be peeled away. This disturbs the on-going vapor equilibration process. Also, after several microscopic inspections, the mylar film can no longer maintain a good seal in all the wells. These problems have inhibited wide use of the MD/24 chamber for routine screening.
In addition, various U.S. patents show and/or disclose related structures.
For example, U.S. Pat. No. 3,107,204 to Brown et al discloses a microbiological testing method and structure therefor. Specifically, the patent discloses a tray having a plurality of wells therein, and a cover for covering the tray. The cover is sealed to the tray around the outer periphery, and importantly, also includes projections which tightly fit within the wells so as to individually seal the same. There is no indication that there is a gaseous seal of the wells. Further, there is a snap-fitting or tight connecting seal between the cover and tray, which would make it difficult to use the same as a crystallization chamber in connection with the vapor diffusion hanging drop method for forming macromolecular crystals.
U.S. Pat. No. 3,165,450 to Scheidt discloses an anaerobic culturing device formed by a shallow dish having partitions which partition the dish into four quadrants. The partitions are of a height lower than the outer wall of the dish. Thus, even when the cover is sealed to the dish, the chambers formed by the partitions are in open gaseous communication with each other. Therefore, this device could not be used to form individual sealed chambers of a crystallization chamber. See also U.S. Pat. No. 3,055,808 to Henderson which is similar and suffers from the same deficiencies.
U.S. Pat. No. 2,561,339 to Chediak discloses a similar arrangement, and it is clear that the wells are in open communication with each other. See also U.S. Pat. No. 4,822,741 to Banes.
U.S. Pat. No. 4,770,856 to Uthemann et al discloses an arrangement in which the tray has a plurality of wells. The tray or plate has a peripheral ledge on which the cover rests. Therefore, this arrangement is similar to the FALCON 3047 MULTIWELL tissue culture plate of Becton-Dickinson and Co., and is deficient for the same reasons for use as a crystallization chamber. See also the prior art description in FIGS. 2 and 3B of U.S. Pat. No. 4,682,891 to de Macario et al.
U.S. Pat. No. 4,012,288 to Lyman et al discloses a tissue culture cluster dish which is similar to the FALCON 3047 MULTIWELL plate by Becton-Dickinson and Co. Although the upper ends of the well walls extend above the upper platform, the lid or cover is supported on the base such that the lower surface of the lid lies vertically above and spaced from the well walls, thereby leaving small gaps.
U.S. Pat. No. 4,599,314 to Shami discloses a multiple vessel specimen tray with a lid for releasably adhering vessel covers. However, the covers are independent and separate for each vessel, that is, there is no common cover for all of the wells.
U.S. Pat. No. 4,599,315 to Terasaki et al discloses a microdroplet test apparatus in which a tray is formed with multiple wells therein. The cover has various rods which project into the wells. However, the rods do not provide a sealing action, and are only used to better optically view the contents of the wells. Further, the wells are in gaseous communication with each other.
U.S. Pat. No. 4,299,921 to Youssef discloses a prolonged incubation microbiological apparatus. However, there is only a single dish with a single chamber.